Prevalence, type of infections and comparative analysis of detection techniques of intestinal parasites in the province of Belgarn, Saudi Arabia

Background The study was conducted to observe the prevalence and type of infection caused by intestinal parasites and to compare the techniques that are available for the detection. Method A total of 112 stool samples were obtained from study participants, and the laboratory examinations were performed at Special Infectious Agents Unit, King Abdulaziz University, Jeddah. One hundred and twelve participants were included in this study. Result The color of positive specimens was mainly brown (86.4%). Stool consistency in infected cases was soft in (64%) samples. There was no statistically significant difference with the physical characteristics of the stool (P > 0.05). In total, 59 of the 112 participants were infected with intestinal parasites, representing 52.7%. Different intestinal protozoa parasites were identified in which Blastocystis hominis (86.4%) was highest. None of the intestinal helminths were detected. Out of the 59 infected cases, single infections were found in (62.7%) samples. The intestinal protozoan parasites in single infections were B. hominis (78.4%), Giardia lamblia (8.1%), and (2.7%) from each Entamoeba histolytica, Cryptosporidium parvum, Entamoeba coli, Endolimax nana, and Chilomastix mesnili. Microscopy, RDTs, and real-time PCR were used for detection and identification of G. lamblia, E. histolytica, and C. parvum. Conclusion The study reported a high rate of intestinal parasitic infections, mainly with B. hominis. There were no statistical differences in parasite detection between the three techniques in detection of the thirteen cases infected with the pathogenic protozoa.


INTRODUCTION
Intestinal parasitic infections (IPIs) are one of the most common infectious diseases, posing health risks in a variety of countries, particularly in developing countries (Shahnazi et al., limited. Real-time polymerase chain reaction (PCR) is a highly accurate technique that can detect intestinal parasites even without symptoms (Ten Hove et al., 2009).
This study was conducted for the first time among all food handlers in Belgarn province, Saudi Arabia, to assess the prevalence, type of intestinal parasites, and compare between the performed detection techniques.

Study area and participants
This study was approved by the research and ethics committee of the Faculty of Applied Medical Sciences at King Abdulaziz University, Jeddah, Saudi Arabia (approval number FAMS-EC2021-08). It was a cross sectional study in which participants were collected from Belgarn province of Saudi Arabia, conducted between April and September 2021. Belgarn is a small province, located in the southwestern part of Saudi Arabia, on mountaintops two thousand feet above sea level. The total area of the province is 1,415 square kilometers, and the population is about 75,000 people.
The total number of food handlers working in the province is 112. All these food handlers were included in the study over the official arrangement and supervision of the Municipality of Belgarn province, without any exclusion criteria.
Simply, a food handler in this study is anyone who works in a food service establishment (restaurants, cafeterias, and coffee shops) and prepares, processes or serves food, or comes into contact with equipment and utensils used to prepare or serve food.
Each participant was provided with a clean labeled plastic container and instructions to collect a stool sample (1-Collect first morning stool directly in the provided labelled clean wide mouth container. 2-Try to fill around a third of the container. 3-The specimen should not be contaminated with water or urine. 4-Close the container tightly. 5-Thoroughly wash hands with soap and water. 6-Place the container in the provided collection bag and keep it in a cool place until obtained from you on the same day).
Each participant signed a consent form as well as filling out a questionnaire to collect data. All collected samples were transported to the microbiology laboratory at Sabt Al-Alaya General Hospital to perform macroscopic examination. For the remaining examinations, samples were transferred to the Special Infectious Agents Unit at King Fahd Medical Research Center, King Abdulaziz University in Jeddah.
In cooperation with the Municipality of Belgarn province, each infected food handler was instructed to visit Sabt Al-Alaya General Hospital for follow up.

Macroscopic and microscopic examination
The stool samples were investigated macroscopically for color, consistency, the existence of blood and mucus, and for any adult worms or proglottids of tapeworms that could be seen in stool.
Two mg of stool was mixed in 1-2 drops of iodine solution to prepare direct smears. Spread evenly on a microscope slide, covered with a cover glass, and scanned on a light microscope with 10× and 40× objective lenses (Garcia et al., 2017).
The sedimentation technique was performed by emulsifying 1-2 g from each stool sample in a 50 ml tube with 10 ml of 10% formal saline solution. The sample was centrifuged at 2,000 rpm for 5 min, then the supernatant was decanted. The sediment was resuspended in 10 ml of 10% formal saline, 3 ml ether was applied, and the mixture was properly shaken before centrifuging for 5 min at 2,000 rpm. The first three layers were removed, and the sediment was investigated under a light microscope after being mixed with two drops of iodine (Garcia et al., 2017).
SDL Quick Trichrome Stain System was used to confirm applicable intestinal protozoan stages. A fecal sample was smeared on a glass slide and allowed to air dry. The slide was dipped four times in 70% ethanol before being immersed in heated trichrome stain for 20 s. Then, the slide was dipped four times in decolorizer and then four times in 90% ethanol. Finally, the smear was dipped four times in xylene, then examined with an oil immersion objective lens (Scientificdevice, 2018).
The modified Kinyoun's stain was used to detect Cryptosporidium. A stool smear was prepared from each sample and allowed to air dry, then fixed with methanol. After that, the smear was stained for 5 min with carbol-fuchsin. Each smear was then washed with tap water, decolorized for 2 min in acid alcohol, washed with tap water, and then counter-stained for 5 min with methylene blue. Finally, the stained smear was washed with tap water, air dried, and examined under the light microscope (Garcia et al., 2017).

Rapid Diagnostic Tests (RDTs)
The CerTest Biotec rapid card was used to detect Cryptosporidium and Giardia, while E. histolytica was detected by Operon kit. For Cryptosporidium and Giardia, the stool collection tube cap was removed, and the stick was used to collect enough sample. To ensure good sample dispersion, the tube was shaken. The CerTest Crypto+Giardia combo card test was opened, and four drops were poured into each circular window. The results were read after 10 min and interpreted based on the appearance of the lines (CerTest, 2021).
For the Operon-E. histolytica test, 1 ml of extraction buffer was placed in a testing tube, and about 50 mg of stool sample was added with a wooden stick and centrifuged for 5 min at 3,000 rpm after properly shaking the test tube. A total of 125 µl of supernatant was added to the round window of the reaction device, 5 min of incubation at room temperature, and the result was read based on the appearance of the lines (Operon, 2018).

Molecular Diagnosis (Real-time PCR)
The extracted DNA was used to detect the presence of three pathogenic intestinal parasites: G. lamblia, E. histolytica, and C. parvum using different primer set (Llewellyn et al., 2016;Won et al., 2016), (Table 1). The QIAamp Fast DNA Stool Mini kit was used for DNA extraction from frozen stool specimens stored at −20 • C. Approximately 180-220 mg or 200 µl of watery sample was taken from the stool sample and placed in a 2 ml microcentrifuge tube that was kept on ice. After that, 1 ml of InhibitEX buffer was added to the sample, and the sample was vortexed for 1 min. 1.2 ml of stool lysate was added to a microcentrifuge tube, and the sample was then centrifuged at full speed for 1 min to pellet feces particles. In a new 2 ml microcentrifuge tube that included 25 µl proteinase K, 600 µl supernatant was Table 1 Real-time PCR primers and probes. This table shows the used primers and probes with realtime PCR. placed, followed by 600 µl Buffer AL and then vortexed for 15 s. The sample was incubated for 10 min in a water bath at 70 • C, then was shaken briefly to eliminate any droplets from the tube cover. A total of 600 µl lysate was properly applied to the QIAamp spin column and was centrifuged at full speed for 1 min. The QIAamp spin column was transferred into a new 2 ml collecting tube. Following that, 500 µl of AW1 buffer was placed to the QIAamp spin column and was centrifuged for 1 min at full speed. After centrifugation, the QIAamp spin column was transferred into a new collecting tube. Following that, 500 µl AW2 buffer was gently placed to the QIAamp spin column and was centrifuged for 3 min at full speed. The QIAamp spin column was then transferred to a new, labeled 1.5 ml microcentrifuge tube, and 200 µl buffer ATE was added immediately onto the QIAamp membrane. Lastly, after 1 min of room temperature incubation, centrifugation at full speed for 1 min was used to elute the DNA. The extracted DNA samples were kept at −20 • C for later use (QIAamp, 2020). For real-time PCR amplification, primer and probe were diluted by adding 10 µl of stock to a new labeled 1.5 microcentrifuge tube, followed by 90 µl of RNase/DNase free water to make a final volume of 100 µl (dilution factor = 1:10). The master mix was made in a 1.5 ml microcentrifuge tube based on the QuantiTect R Probe PCR kit guidelines, then was vortexed thoroughly, and finally was centrifuged at full speed. The reaction mix was then transferred in the required amount (15.5 µl) to a standard PCR plate, and 4.5 µl of pure DNA was placed to each well ( Table 2). As a negative control, a PCR reaction with no DNA template (just RNase/DNase free water) was performed for every run. The plate was centrifuged for 1 min to mix the components. To begin the cycling program, the plate was placed in an Applied Biosystems 7500 Fast cycler, and the condition was set in accordance with the QuantiTect R Probe PCR kit's guidelines (QuantiTect, 2011). The Applied Biosystems 7500 Fast System was used to analyze the data, which computed the Ct value (threshold cycle number) for positive gene amplification in real-time-cycler number.

Statistical analysis
The data collected was analyzed using the Statistical Package for the Social Sciences (the IBM SPSS R version 25 statistical software). The descriptive analysis was used to evaluate demographic data and categorical variables. All reported P values were two-tailed and P < 0.05 is considered significant.

RESULTS
One hundred and twelve participants ranging in age from 20 to 65 years old (33.5 ± 9.2) were included in this study. Information on socio-demographic status, nationality, level of education, symptoms and hygienic habits is being presented as part of a separate study and is not included here. The color of positive specimens ranged from brown (51, 86.4%) to dark brown (8, 13.6%). Stool consistency in infected cases including pathogenic and nonpathogenic parasites was soft in 38 samples, formed in 17, and loose in four, whereas consistency in uninfected cases was soft in 42 samples, formed in seven, and loose in four (Fig. 1). There were no adult worms or proglottids of tapeworms in any of the investigated samples. There was no statistically significant difference with the physical characteristics of the stool. In total, 59 of the 112 participants were infected with intestinal parasites, representing 52.7%. Using microscopic examinations, different intestinal protozoa parasites were identified such as: B. hominis 51 (86.4%), Entamoeba coli 13 (22%), E. nana 11 (18.6%), G. lamblia 6 (10.2%), E. histolytica 6 (10.2%), C. mesnili 4 (6.8%), Iodamoeba buetschlii 1 (1.7%), and C. parvum 1 (1.7%). Using microscopy, no eggs or other diagnostic stages of intestinal helminths were detected.
Pathogenic intestinal protozoa G. lamblia, E. histolytica, and C. parvum were identified using microscopy, RDTs, and real-time PCR. There were no differences in parasite detection between the three techniques for G. lamblia (6, 10.2%) and C. parvum (1, 1.7%). For E. histolytica (6, 10.2%), RDTs detected parasites in only three samples, whereas real-time PCR identified E. histolytica in all six samples. There was no statistically significant difference between the techniques used (P = 0.53) for detection of the pathogenic protozoa (Table 4). The infected samples with G.lamblia (n = 6), E. histolytica (n = 6), and Cryptosporidium (n = 1) are not the same samples.
Direct methods, concentration methods, permanent stains, RDTs, and real-time PCR were all used in this study. Iodine is used in direct smear examination to detect the details of diagnostic features of different parasites. As a result, direct smear examination is regarded as the most suitable and simple method of detecting intestinal parasites. On the other hand, direct smear examination may produce false negative results in cases of low infection levels, requiring the use of more accurate methods. Several concentration methods are Table 3 The prevalence of the detected parasites in each infection type, using microscopy. The prevalence of all detected parasites (pathogenic and nopathopgenic) in each infection type, using microscopic examination techniques.

Type of infection
No.
Single infection (  commonly used to improve the possibility of detecting parasitic diagnostic stages. One of the most useful concentration techniques for detecting cysts, ova, and larvae in large quantities of stool is the Ritchie sedimentation method. The diagnostic stages are killed and preserved using formal saline, while diethyl ether separates the undesirable debris at the top of the tube and concentrates all the diagnostic stages at the bottom. On the other hand, trophozoites are undetectable due to their adherence to debris, and the chemicals required in this method are carcinogenic and flammable. Permanent trichrome and modified Kinyoun's stains were used in this study. Trichrome is regarded as the most suitable stain for detecting cyst and trophozoite morphological characteristics. Because wet mount examinations could miss small protozoa, trophozoites quickly perish, and immediate processing of samples is difficult, staining methods are the best choice. The modified Kinyoun's stain can be used to identify coccidian oocysts such as Cryptosporidium, Cystoisospora, and Cyclospora from fresh or preserved stool samples that are difficult to identify using routine stains (Wakid, 2006;Zaglool et al., 2011;Taha, Soliman & Banjar, 2013;Wakid, 2020). RDTs use monoclonal antibodies inserted into a device to identify one or more antigens. Two commercial kits were used in this study: the CerTest Crypto+Giardia combo card test and the Operon-Entamoeba test. These chromatographic immunoassay kits for detecting three primary protozoan-induced diarrheal disorders. Antigen detection techniques are simple to use and do not require the assistance of a qualified microscopist. Most kits require fresh or preserved stool specimens for antigen detection methods (Wakid, 2006;Zaglool et al., 2011;Taha, Soliman & Banjar, 2013;Wakid, 2020).
As a molecular technique, real-time PCR was used in this study to confirm isolated parasites. For detecting parasites in stool specimens, real-time PCR was found to be superior to microscopic investigation. These methods have the advantage of being able to identify low parasite levels. On the other hand, the high risk of contamination and the high cost of reagents are significant disadvantages. In the present study, 59 participants were infected with either a single parasite or multiple parasites. This is consistent with prior studies in Saudi Arabia, which found that infection rate with a single parasite was higher than multiple parasites (Wakid, 2006;Zaglool et al., 2011;Taha, Soliman & Banjar, 2013;Wakid, 2020). All detected intestinal parasites in our study were protozoa. According to previous studies in Saudi Arabia, protozoa were the most frequently detected parasites (Wakid, 2006;Zaglool et al., 2011;Taha, Soliman & Banjar, 2013;Wakid, 2020). The reason for that is the simple route of protozoa transmission. Additionally, the high prevalence of protozoa can be explained by their high proliferation, ability to form cysts, and resistance to environmental factors (Heydari-Hengami et al., 2018). Pathogenic and opportunistic protozoan parasites were identified in approximately 22.1% of the isolated organisms.
These protozoa were G. lamblia (10.2%), E. histolytica (10.2%), and C. parvum (1.7%). There were no abdominal symptoms in 84.6% of those infected with these protozoa. Asymptomatic infected food handlers pose a significant risk of spreading the infection to their coworkers and customers. In comparison to other studies, the current study found that parasitic infection was more prevalent in Belgarn province. The study also revealed that non-pathogenic parasites accounted for 49.1%, including E. coli (22%), E. nana (18.6%), C.